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CN116555290B - Method for improving yield and resistance of indica rice variety by OsPIL1 gene and application thereof - Google Patents

Method for improving yield and resistance of indica rice variety by OsPIL1 gene and application thereof Download PDF

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CN116555290B
CN116555290B CN202310804629.0A CN202310804629A CN116555290B CN 116555290 B CN116555290 B CN 116555290B CN 202310804629 A CN202310804629 A CN 202310804629A CN 116555290 B CN116555290 B CN 116555290B
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ospil1
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杨静
赵添琦
唐萍
苏顺雨
刘祟兰
钟爰爰
左如斌
罗琼
李永杰
晁钟毅
杨蕊熔
刘娜
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Yunnan Agricultural University
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Abstract

The invention relates to the field of biotechnology, in particular to a biological preparation methodOsPIL1The gene is applied in the method for improving the yield and the resistance of indica rice variety,OsPIL1the gene sequence is shown as SEQ ID No.1, willOsPIL1When the expression level of the gene in the indica rice variety reaches 1.1 times of the expression level of wild rice by using an agrobacterium-mediated genetic transformation method, the growth, grain length, yield and resistance of the rice can be obviously improvedOsPIL1Genes provide references in improving the growth, yield and resistance of other crops. The method is toOsPIL1The gene is over-expressed in the indica rice variety, so that the growth quantity, grain length, yield and resistance of the indica rice variety are improved, and the method can be also used for improving the indica rice variety or breeding new varieties.

Description

OsPIL1Method and application of gene in improving yield and resistance of indica rice variety
Technical Field
The invention relates to the field of biotechnology, in particular to a biological preparation methodOsPIL1The gene can be used in the method for improving the yield and resistance of indica rice.
Background
Rice is one of the main food crops in most countries around the world. The occurrence and popularity of rice diseases seriously threatens global rice yield and quality, and disease management measures such as chemical control and disease-resistant varieties have a certain effect on reducing rice yield loss, however, chemical control faces the problems of environmental pollution, rice pesticide residues and the like affecting rice quality. The disease-resistant variety is difficult to achieve the purposes of high yield, high quality and disease resistance because of the negative correlation among agronomic traits such as crop growth, yield, quality and resistance. Thus, how to coordinate the balance among growth, yield and resistance in existing rice disease-resistant breeding is a formidable challenge, but cultivation of high-yield, high-quality and high-resistance rice varieties is an optimal rice disease management strategy.
Many broad-spectrum resistance genes or genotypes with greater immunity and no yield loss are being integrated into crop varieties, improving crop resistance without affecting crop growth or yield. For example, the rice blast CP protein MoSM1 is over expressed in rice, so that the rice resistance to rice blast and xanthomonas is enhanced, and the rice growth and yield are not negatively affectedInfluence. NLR receptor genePigmR6The over-expressed transgenic rice lines have improved pestilence resistance and likewise have no negative effect on rice growth and yield. The rice carrying the Xa4+xa5+Xa21 genotype had high resistance to 18 Xanthomonas oryzae strains from Korea, and had no negative effect on rice growth and yield. Overexpression of the P450 protein BSR2 (BROADSPECTRUM RESISTANCE) in Arabidopsis and rice enhances disease resistance of Arabidopsis and rice against Rhizoctonia solani, while slightly slowing the growth rate of rice, but increasing the grain of rice. The ideal strain type gene IPA1 (IDEAL PLANT ARCHITECTURE 1) transgenic rice strain has enhanced pestilence resistance and has no negative effect on rice growth and yield. Mutants of the tetrapeptide repeat (TPR) domain RNA binding protein BSR-K1 exhibit broad spectrum resistance and key agronomic traits remain good. However, the introduction or integration of these genes or genotypes into crop varieties increases crop resistance, but has no effect on crop growth or yield.
Disclosure of Invention
The invention aims to provide a method for manufacturing the LED display deviceOsPIL1The gene can be used in the method for improving the yield and resistance of indica rice.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention uses the rice photosensitive pigment interaction factorsOsPIL1The gene is used as the object, the genome DNA is extracted from the paddy rice moon valley and is reversely transcribed into cDNA, and the PCR technology is used for amplifyingOsPIL1The CDS region sequence 1266bp coding region of the gene, the gene sequence is shown as SQE ID No.1, and the coded amino acid sequence is shown as SQE ID No. 2. By constructingOsPIL1The gene over-expression vector is introduced into the moon of the normal indica rice variety by adopting the agrobacterium LBA4404 mediated genetic transformation method to obtainOsPIL1Plants with over-expressed genes, whenOsPIL1When the expression level of the gene exceeds 1.1 times, the growth level, grain length, yield and resistance of the gene are obviously improved compared with those of the wild rice moon valley. Editing knockout of moon valleys by CRISPR/Cas9 technologyOsPIL1Genes are obtainedOsPIL1Knock-out plants of knock-out rice plants with significantly lower growth, grain length, yield and resistanceIn the moon valley of wild rice. These results indicate that by increasingOsPIL1The expression level of the gene can promote the increase of the growth level, grain length and yield of rice and improve the resistance of the rice.
Accordingly, the invention is claimedOsPIL1The following uses of the gene:
as shown in SEQ ID No.1OsPIL1The application of the gene or the coded protein in improving the growth of indica rice varieties of rice.
As shown in SEQ ID No.1OsPIL1The application of the gene or the coded protein in improving the grain length of the indica rice variety of the rice.
As shown in SEQ ID No.1OsPIL1The application of the gene or the coded protein in improving the yield of the indica rice variety of the rice.
As shown in SEQ ID No.1OsPIL1The gene or the coded protein is applied to improving the disease resistance of the indica rice variety.
As shown in SEQ ID No.1OsPIL1The gene or the coded protein is applied to improving the rice variety improvement or breeding.
The invention also claims a polypeptide shown in SEQ ID No.1OsPIL1The method for improving the growth quantity, grain length, yield and disease resistance of the indica rice variety by using the gene or the coded protein comprises the following steps:
(1) Extracting genome DNA from wild rice moon valley;
(2) Amplifying the target gene by PCR technologyOsPIL1The target geneOsPIL1The sequence is shown as SEQ ID No. 1;
(3) Genes are addedOsPIL1The fragment is connected to an expression vector to obtain over-expressionOsPIL1Plasmid pBWA (V) HS-OsPIL1-osgfp of target gene;
(4) Introducing a plasmid pBWA (V) HS-OsPIL1-osgfp into Agrobacterium;
(5) The agrobacterium containing plasmid pBWA (V) HS-OsPIL1-osgfp is co-cultured with indica rice variety rice plant to obtain the rice plant system with positive correlation of growth quantity, grain length, yield and disease resistance.
Further, in the step (2), the target gene is amplifiedOsPIL1The primer sequences of (2) areOsPIL1-F andOsPIL1-R：
OsPIL1-F:5¢-AACACGGGGACTTTGCAACATGGATGGCAATGCGAGATCGGCGG-3¢，
OsPIL1-R:5¢-TCCTCGCCCTTCACGATACAAATTCCATCAGAGGTTGGTGGTTGT-3¢。
the method is toOsPIL1The target gene is over-expressed in the indica rice variety, so that the growth quantity, grain length, yield and resistance of the indica rice variety are improved, and the method can also be used for improving the indica rice variety or breeding new varieties and also belongs to the protection scope of the invention.
The beneficial technical effects of the invention are as follows: the invention overcomes the defect that the existing gene or genotype is integrated into the crop variety to only improve the resistance, and the photosensitive pigment is interacted with the class factorOsPIL1) The introduction of rice obviously improves the growth, yield and resistance of the rice, and can be a photosensitive pigment interaction factorOsPIL1) Providing a reference in improving the growth, yield and resistance of other crops, furthermore, the method willOsPIL1The target gene is over-expressed in the indica rice variety, so that the growth quantity, yield and resistance of the indica rice variety are improved, and the method can also be used for improving the indica rice variety or breeding new varieties.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a drawing of rice seeds, roots, stems and leaves of example 2OsPIL1Gene expression level;
FIG. 2 is rice of example 3OsPIL1A graph of the results of the effect of gene overexpression on coleoptile length;
FIG. 3 is rice of example 3OsPIL1Gene overexpression on rice plantsA highly influenced results plot;
FIG. 4 shows rice in example 3OsPIL1A result graph of the influence of the gene overexpression on the internode length of the rice;
FIG. 5 shows rice in example 3OsPIL1A result graph of the influence of the gene overexpression on the length of the rice ears;
FIG. 6 is rice of example 3OsPIL1A result graph of the influence of the gene overexpression on the length of the rice roots;
FIG. 7 shows rice in example 3OsPIL1A result plot of the effect of gene overexpression on rice grain weight and grain length;
FIG. 8 is rice in example 4OsPIL1A result graph of the influence of the gene overexpression on the disease index of the rice;
FIG. 9 is rice of example 4OsPIL1A result graph of the effect of gene overexpression on the length of the disease spots and the disease spot biomass of the rice;
FIG. 10 is rice in example 5OsPIL1Results of gene overexpression effect on rice growth, yield and resistance related gene expression.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are all conventional and can be carried out according to the described recombinant techniques (see molecular cloning, laboratory manual, 2 nd edition, cold spring harbor laboratory Press, cold spring harbor, N.Y.); the materials, reagents, and the like used are all commercially available.
Example 1
OsPIL1The construction of the transgenic plant with the over-expressed gene comprises the following steps:
1. genomic DNA was extracted from wild rice moon valleys:
2. amplifying key gene by PCR techniqueOsPIL11266bp coding region (shown in SEQ ID No. 1) using restriction site primers:
F:5¢-AACACGGGGACTTTGCAACATGGATGGCAATGCGAGATCGGCGG-3¢(SEQ ID No.3)，
R:5¢-TCCTCGCCCTTCACGATACAAATTCCATCAGAGGTTGGTGGTTGT-3¢(SEQ ID No.4)。
electrophoresis on a 1% agarose gel, 5v/cm voltage, 20 minutes, will: the 1266bp electrophoresis fragment is cut out under an ultraviolet lamp, put in a system for sol recovery, the recovery procedure is shown in the specification of a kit of a specific manufacturer, the DNA is recovered by dissolving with water with the total volume of 40 mu L (the recovered product is marked as rDNAG 1), and the DNA is recombined with a carrier after detection.
3. Enzyme cutting of carrier
The vector pBWA (V) HS-OsPIL1-osgfp was purified using a PCR purification kit (the purification product was labeled pBWA (V) HS-ccdb-osgfp (D)) for the next step of in vitro or in vivo recombination reaction.
The carrier construction mainly adopts homologous recombination and golden gate seamless cloning methods, and two ends of the target fragment after construction do not contain enzyme cutting sites, so that the target gene fragment and the carrier skeleton fragment cannot be obtained through double enzyme cutting. However, to ensure the accuracy of the recombinant plasmid, we used EcoRV endonuclease to cleave the recombinant plasmid outside of the sequencing, and verified whether the actual fragment size of the recombinant plasmid was consistent with the theoretical value. Therefore, the restriction enzyme verification graph is not a double restriction enzyme verification graph, and is an accurate quality control graph of the recombinant plasmid.
4. Recombination reactions, converting the ligation products into competent cells.
5. Transformation
Transformation of 5-10. Mu.L ligation products into E.coli competent cells (see E.coli competent transformation standard method) were transformed into (calicheamicin) resistant plates and incubated at 37℃for 12 hours for plaque PCR identification.
6. Plaque PCR identification
10 plaques were picked and subjected to 1.5ml EP tube-connected and PCR identification simultaneously, primers: pBWA (V) HS-ccdb-osgfp identifies primer 35seq (G), noseq (G).
7. Agrobacterium transformation of rice
（1）OsPIL1Transforming rice callus by using an overexpression plasmid: by constructing the aboveOsPIL1The overexpression plasmid was transformed with Agrobacterium tumefaciens (LBA 4404) and then this was transformed withOsPIL1The agrobacterium tumefaciens strain overexpressing the plasmid was cultured on YEP medium containing rifampicin and hygromycin at 28 ℃ at 250r/min overnight. Taking 300ul of overnight culture solution in 3ml of YEP liquid culture medium containing corresponding antibiotics, and shake culturing at 28deg.C under 250r/min until bacterial solution reaches OD 600 =0.5, i.e. the agrobacterium tumefaciens dip is prepared and can be used for infection.
Selecting rice granular embryogenic callus with good growth state, properly clamping the rice granular embryogenic callus with sterile forceps to create a wound, then soaking the wound in bacterial liquid for 10-30 min, wherein the bacterial liquid can be soaked for a period of time longer than a period of time when the bacterial liquid is not high in concentration for a few minutes, and the wound is shaken from time to time when the bacterial liquid is soaked, and then placing the wound into a plate with filter paper to suck excessive bacterial liquid.
The callus of the blotted fungus liquid is placed on NBco solid culture medium, 1 layer of paper is placed on the co-culture medium, and the callus is placed on filter paper. Dark culture is carried out for 2-3 days at 25-27 ℃ until a small amount of bacterial plaque appears on the callus.
(2) Degerming and screening of the callus: the co-cultured callus is placed in a wide-mouth bottle, washed to be clear by sterile water, soaked in 500mg/L NBco liquid medium containing Cef (cephalomum light-yellow) and oscillated for 30-60min on a shaking table, the liquid is discarded, the callus is sucked to be dry by sterile filter paper or blown dry on an ultra-clean workbench and then placed on a screen of culture medium for dark culture for three weeks, and then transferred to a second screen of culture medium for dark culture for three weeks, and the temperature is controlled at 25-27 ℃.
(3) Differentiation and rooting of resistant calli: and (3) inoculating the newly grown resistant callus after the twice screening to a pre-differentiation culture medium, culturing in dark for 10 days, transferring to the differentiation culture medium for illumination culture, and illuminating for 12 hours each day by using a fluorescent lamp, wherein the temperature is controlled at 25-27 ℃ for 1-2 months, so as to obtain seedlings with the height of 2-3 cm.
Transferring seedling to rooting culture medium, culturing, taking out when root grows to 2-2.5cm high, cleaning root culture medium, transplanting into field soil, and transferring to outdoor environment suitable for rice after greenhouse culture for a period of time.
Example 2
OsPIL1Expression levels in rice seeds, roots, stems and leaves
qRT-PCR detectionOsPIL1At the position ofOsPIL1The expression levels in the seeds, roots, stems and leaves of the transgenic rice were overexpressed, and the results showed that, compared with the wild-type rice (WT),OsPIL1the expression level in the rice seeds was 1.7 times that of the wild type rice (WT),OsPIL1the expression levels in the roots, stems and leaves of rice at 30 days were 2.87-fold, 1.09-fold and 1.28-fold, respectively, while the expression levels in the roots, stems and leaves of rice at 60 days were 2.1-fold, 6.0-fold and 1.59-fold, respectively. Thus, the first and second substrates are bonded together,OsPIL1the expression level in the seeds, roots, stems and leaves of rice was 1.09 times or more higher than that in wild type rice, and the growth, yield and resistance of rice could be promoted (FIG. 1).
Example 3
OsPIL1Promoting phenotype and grain size of rice plants
(1) Phenotype of rice plants: over-expressed rice lines were observed and countedOsPIL1The phenotypes of OE (# 1, #2, #6, #1, #2, #6 are three lines of OsPIL1 transgenic overexpressing rice) including plant height, stem length, coleoptile length, ear length, root length, hundred grain weight and seed vertical length, and as a result, three were foundOsPIL1The coleoptile length of the over-expressed transgenic rice was significantly longer than that of WT (figure 2),OsPIL1the height of the overexpressing plants was significantly higher than that of wild-type rice (FIG. 3), and the length of each internode of the adult OsPIL1 and WT plants was further measured and foundOsPIL1The length of each internode of the over-expressed plants was significantly longer than that of WT (figure 4),OsPIL1the spike length of the overexpressing plants was significantly longer than that of WT (figure 5),OsPIL1the root length of the overexpressing plants was significantly longer than that of WT (fig. 6), the hundred grain weights and grain lengths of OsPIL1 overexpression were significantly higher than that of WT (fig. 7).
Example 4
OsPIL1Identification of pestilence resistance of over-expressed transgenic rice
(1) Rice blast fungus inoculationOsPIL1Observation and statistics of rice blast symptom of over-expression transgenic rice
Rice blast fungus inoculationOsPIL1The over-expression transgenic rice was spray inoculated with Pyricularia oryzae (95234I-1 b), and the disease index was investigated after inoculation with 144 hpi. As a result, it was found that, compared with the WT strain,OsPIL1the disease index of the over-expressed transgenic lines was significantly reduced (fig. 8). For a pair ofOsPIL1Over-expressed transgenic Rice punch inoculated with Pyricularia oryzae (95234I-1 b), inoculated with 144hpi and measured for lesion length and lesion biomass, foundOsPIL1Both the lesion length and the lesion biomass of the over-expressed transgenic lines were significantly lower than that of WT (fig. 9).
Example 5
RNA-seq resolutionOsPIL1Transcriptional control of genes related to rice growth, yield and resistance
(1) RNA-seq identificationOsPIL1Gene related to regulation and control of rice growth, yield and resistance
RNA-seq screening to Pyricularia oryzae inoculationOsPIL1Gene related to root growth in over-expression transgenic riceOsRPC53、BRX、OsBRL1、OsPIN10a、OsAAO2The method comprises the steps of carrying out a first treatment on the surface of the Genes related to stem growthONI3、BRXThe method comprises the steps of carrying out a first treatment on the surface of the Gene related to tillering numberOscZOG1The method comprises the steps of carrying out a first treatment on the surface of the Chlorophyll synthesis-related gene peptidyl-tRNA hydrolase (LOC_Os 03g 22610); genes related to leaf angleOsIAA12、SLG、OsBC1、OsBUL1Other genes associated with growth: ankyrin gene (LOC_Os 09g 03750), protein-expanding precursor family gene, zinc finger protein family gene、OsRopGEF10、RALFL26Isogenic, yield-related genes (RGH 1A family genes, grain maturation quality-related genes)LTPL26Genes related to grain sizeOsAK3Plant organ size genesARGOSDelay flowering time, increase plant height and increase grain yieldOsCOL16Signal transduction related calmodulin binding protein family gene in stress, resistance related genes (membrane protein family genes of OsJMT1 and CBS domains),OsDof28OsWRKY 89) isogenic upregulation。
(2) RT-qPCR verifies the expression of the gene in rice plants, grains and rice plants infected by rice blast fungus
We selected 11 genesOsRPC53(LOC_Os04 g 32350), a expansin precursor (LOC_Os02g 44108), a calmodulin binding protein (LOC_Os11g 44600), an anchoring protein (LOC_Os09 g 03750), zeatin O-glucosyltransferase 1 (OscZOG 1),OsJMT1、OsAAO2、RGH1A、LTPL26、RALFL26、OsCOL16RT-qPCR assays were performed to verify their presence in wild type and in wild typeOsPIL1Cereal grains, roots, stems, leaves and expression of the overexpressed lines infected with Pyricularia oryzae for 48h and 72 h. As a result, it was found that, compared with the wild type,OsRPC53down-regulation of expression in grains, leaves and infected rice plants, but inOsPIL1Upregulation of expression in roots and stems of OE; anchor protein (LOC_Os 09g 03750) andOscZOG1expression was down-regulated in grains, roots and stems, whereas expression was significantly up-regulated in leaves and infected OsPIL1 overexpressing seedlings.OsJMT1Only infected withOsPIL1Up-regulation of expression in the over-expressed plants was not detected in the grain, roots, stems and leaves.OsAAO2In grains, roots, stems and infested plantsOsPIL1Down-expression in overexpressing plants, whileOsPIL1Up-regulation of expression in leaves of over-expressed plants.RGH1A、RALFL26AndOsCOL16expression is significantly up-regulated in grains, roots, stems and leaves, but is infestedOsPIL1No detection was observed in the overexpressing plants.LTPL26In cereal grains and infested plantsOsPIL1Expression is up-regulated in overexpressing plants, but down-regulated in roots, stems and leaves. Gene of calmodulin binding protein (LOC_Os11g 44600) in grains, roots, stems, leaves and infectedOsPIL1Upregulation of expression in the overexpressing plants (fig. 10).
Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be encompassed by the claims.

Claims (3)

1. As shown in SEQ ID No.1OsPIL1Use of a gene or a protein encoded thereby for increasing the yield of indica rice varieties, characterized in that the application steps comprise:
(1) Extracting genome DNA from wild rice moon valley;
(2) Amplifying the target gene by PCR technologyOsPIL1The target geneOsPIL1The sequence is shown as SEQ ID No. 1;
(3) Genes are addedOsPIL1The fragment is connected to an expression vector to obtain over-expressionOsPIL1Plasmid pBWA (V) HS-OsPIL1-osgfp of target gene;
(4) Introducing a plasmid pBWA (V) HS-OsPIL1-osgfp into Agrobacterium;
(5) The agrobacterium containing plasmid pBWA (V) HS-OsPIL1-osgfp is co-cultured with rice strain of indica rice variety to obtain rice strain with improved yield.
2. The use according to claim 1, wherein in step (2), the primer sequences for amplifying the desired gene OsPIL1 are OsPIL1-F and OsPIL1-R:
OsPIL1-F:5¢-AACACGGGGACTTTGCAACATGGATGGCAATGCGAGATCGGCGG-3¢，
OsPIL1-R:5¢-TCCTCGCCCTTCACGATACAAATTCCATCAGAGGTTGGTGGTTGT-3¢。
3. as shown in SEQ ID No.1OsPIL1The application of the gene or the coded protein in the improvement or the breeding of indica rice varieties is characterized in that the application refers to the improvement of the yield of the indica rice varieties in the improvement or the breeding of the indica rice varieties.
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